Institute of Toxicology, Medical Center of the University Mainz, Obere Zahlbacher Str 67, D-55131 Mainz, Germany.
Mol Pharmacol. 2010 Nov;78(5):943-51. doi: 10.1124/mol.110.066076. Epub 2010 Aug 20.
Methylating agents are first-line therapeutics for gliomas and malignant melanomas. They attack DNA at various sites, and both O(6)-methylguanine and N-methylated base adducts contribute to the killing response. The mechanism of cellular defense against these agents primarily involves O(6)-methylguanine-DNA methyltransferase (MGMT) and base excision repair (BER). Here, we determined whether a key protein involved in DNA double-strand break (DSB) recognition and signaling, nibrin (NBN alias NBS-1), plays a role in the cellular defense against methylating agents. Comparing NBN mutated fibroblasts and lymphoblastoid cells from patients suffering from Nijmegen breakage syndrome, we show that NBN mutants are clearly more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and temozolomide than the corresponding wild-type cells. Hypersensitivity was due to the induction of both apoptosis and necrosis. The mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 were expressed at a similar level in the cell lines and BER was not affected by NBN mutation. Because MGMT expression abrogated the hypersensitivity of NBN mutated cells, we conclude that O(6)-methylguanine-derived lesions are responsible for triggering the response. Down-regulation of NBN in melanoma cells by small interfering RNA rendered them more sensitive to temozolomide, suggesting that NBN is a novel modulator of temozolomide sensitivity. Because NBN is part of the MRN complex, which recognizes DSBs, the data strongly indicate that MRN is critically involved in DSB processing after O(6)-methylguanine induction. The data provide first evidence that NBN is involved in the cellular defense against O(6)-methylguanine-inducing agents such as temozolomide and identify NBN as a critical target of methylating anticancer drug resistance.
甲基化试剂是神经胶质瘤和恶性黑色素瘤的一线治疗药物。它们在多个位点攻击 DNA，O(6)-甲基鸟嘌呤和 N-甲基碱基加合物都有助于杀伤反应。细胞对这些试剂的防御机制主要涉及 O(6)-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）和碱基切除修复（BER）。在这里，我们确定了参与 DNA 双链断裂（DSB）识别和信号转导的关键蛋白 nibrin（NBN 别名 NBS-1）是否在细胞对甲基化试剂的防御中发挥作用。比较患有尼曼匹克破碎综合征的患者的 NBN 突变成纤维细胞和淋巴母细胞系，我们发现 NBN 突变体对 N-甲基-N'-硝基-N-亚硝基胍和替莫唑胺的敏感性明显高于相应的野生型细胞。敏感性增加是由于凋亡和坏死的诱导。错配修复蛋白 MSH2、MSH6、MLH1 和 PMS2 在细胞系中的表达水平相似，BER 不受 NBN 突变的影响。由于 MGMT 表达消除了 NBN 突变细胞的超敏性，我们得出结论，O(6)-甲基鸟嘌呤衍生的损伤负责触发反应。用小干扰 RNA 下调黑素瘤细胞中的 NBN 使它们对替莫唑胺更敏感，这表明 NBN 是替莫唑胺敏感性的一种新的调节剂。由于 NBN 是识别 DSB 的 MRN 复合物的一部分，数据强烈表明，MRN 在后 O(6)-甲基鸟嘌呤诱导的 DSB 处理中起着关键作用。这些数据首次证明 NBN 参与了对替莫唑胺等 O(6)-甲基鸟嘌呤诱导剂的细胞防御，并将 NBN 确定为甲基化抗癌药物耐药性的关键靶标。
